Proteome Science BioMed Central©rcio_2006_1027.pdf · Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany and 3Gerência de Animais Peçonhentos

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Open AcceResearchOntogenetic variations in the venom proteome of the Amazonian snake Bothrops atroxRafael AP Guércio1, Anna Shevchenko2, Andrej Shevchenko2, Jorge L López-Lozano3, Jaime Paba1, Marcelo V Sousa1 and Carlos AO Ricart*1
Address: 1Brazilian Center for Protein Research, Department of Cell Biology, University of Brasilia, Brasília, 70910-900- DF, Brazil, 2Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany and 3Gerência de Animais Peçonhentos – Fundação de Medicina Tropical do Amazonas, Av. Pedro Teixeira 25, 69040-000 Manaus, AM, Brazil

Email: Rafael AP Guércio - [email protected]; Anna Shevchenko - [email protected]; Andrej Shevchenko - [email protected]; Jorge L López-Lozano - [email protected]; Jaime Paba - [email protected]; Marcelo V Sousa - [email protected]; Carlos AO Ricart* - [email protected]

* Corresponding author

AbstractBackground: Bothrops atrox is responsible for the majority of snakebite accidents in the BrazilianAmazon region. Previous studies have demonstrated that the biological and pharmacologicalactivities of B. atrox venom alter with the age of the animal. Here, we present a comparativeproteome analysis of B. atrox venom collected from specimens of three different stages ofmaturation: juveniles, sub-adults and adults.

Results: Optimized conditions for two-dimensional gel electrophoresis (2-DE) of pooled venomsamples were achieved using immobilized pH gradient (IPG) gels of non-linear 3–10 pH rangeduring the isoelectric focusing step and 10–20% gradient polyacrylamide gels in the seconddimension. Software-assisted analysis of the 2-DE gels images demonstrated differences in thenumber and intensity of spots in juvenile, sub-adult and adult venoms. Although peptide massfingerprinting (PMF) failed to identify even a minor fraction of spots, it allowed us to group spotsthat displayed similar peptide maps. The spots were subjected to a combination of tandem massspectrometry and Mascot and MS BLAST database searches that identified several classes ofproteins, including metalloproteinases, serine proteinases, lectins, phospholipases A2, L-aminooxidases, nerve growth factors, vascular endothelial growth factors and cysteine-rich secretoryproteins.

Conclusion: The analysis of B. atrox samples from specimens of different ages by 2-DE and massspectrometry suggested that venom proteome alters upon ontogenetic development. Weidentified stage specific and differentially expressed polypeptides that may be responsible for theactivities of the venom in each developmental stage. The results provide insight into the molecularbasis of the relation between symptomatology of snakebite accidents in humans and the venomcomposition. Our findings underscore the importance of the use of venoms from individualspecimen at various stages of maturation for the production of antivenoms.

Published: 11 May 2006

Proteome Science 2006, 4:11 doi:10.1186/1477-5956-4-11

Received: 14 December 2005Accepted: 11 May 2006

This article is available from: http://www.proteomesci.com/content/4/1/11

© 2006 Guércio et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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BackgroundThe genus Bothrops (family Viperidae) comprises severalspecies of pit vipers inhabiting the American continentfrom Mexico to Argentina [1]. Bothrops atrox species areresponsible for the majority of snakebite accidents in theBrazilian Amazon region [2]. In humans, Bothrops atroxenvenomation causes local effects such as swelling, localhemorrhage and necrosis besides systemic effects, includ-ing alterations in blood coagulation and various types ofbleeding distant from the bite site [3]. Perturbed bloodhemostasis and thrombosis are largely caused by protein-ases, especially metallo- and serine- proteinases that arethe major components of Bothrops snake venoms [4].

Among other factors, the composition of snake venoms isaffected by the age of the animals. A comparative study ofthe proteinase activity and protein profiles of venomsfrom juvenile, sub-adult and adult Bothrops atrox speci-mens captured in the Brazilian Amazon rain forest waspreviously reported [2]. López-Lozano et al demonstratedthat venoms from juveniles and sub-adults displayedhigher human plasma clotting activity compared to adultvenoms. In addition, SDS-PAGE and HPLC venom pro-tein profiles varied among the three developmental stagesanalyzed. Two proteins of 23 kDa and 50 kDa, respec-tively, that were present in higher amounts in adult ven-oms, were identified as metalloproteinases.

An independent study of B. atrox specimens from theColombian Amazon rain forest showed that venoms ofnewborn and juvenile specimen caused higher lethalityand possessed higher hemorrhagic and coagulant activi-ties, than adult venoms. The differences in activity wereattributed to the increased amount of high molecularmass proteins, probably also metalloproteinases [5].

Taken together, these and other published evidence indi-cated that changes in the venom proteome during ontoge-netic development can influence its biological activity.Here we report a comparative proteome analysis of B.atrox venoms from juvenile, sub-adult and adult speci-mens that identified proteins whose differential expres-sion during ontogenetic development may be correlatedto the previously reported properties of the venom [2,5].

ResultsIn order to optimize 2-DE separation of B. atrox venomproteins, linear and non-linear 3–10 pH gradients weretested in the isoelectric focusing (IEF) step. The non-lineargradient, developed to improve resolution of acidic pro-teins, provided better resolution of spots than linear pHgradient since many spots consisted of polypeptides dis-playing isoelectric points (pI) between 4 and 7 (data notshown). Two types of electrophoresis equipment – Multi-phor II and IPGphor from GE Healthcare- were tested for

the IEF step and both provided similar 2-DE maps (datanot shown). On the other hand, for the second dimen-sion, gradient gels (10–20% T) provided better 2-DEmaps than 12% T gels, especially for proteins with molec-ular masses around 14 kDa (data not shown).

Patterns of protein spots visualized by silver staining weredifferent between pooled venom samples from juvenile,sub-adult and adult B. atrox (Fig. 1). Their computer-assisted image analyses detected 110 spots in the gels fromjuveniles, 101 in sub-adults and 86 in adult venoms.Among the detected spots, 44 were found specifically injuveniles, 22 in sub-adults and 22 in adults. Image analy-sis also pinpointed substantial differences in the relativeabundance of several spots matched at all three images(Table 1).

The identification of proteins was initially attempted byN-terminal sequencing of the proteins blotted to a PVDFmembrane using Edman degradation. Because of the rela-tively low sensitivity of Edman degradation reliable pep-tide sequences were retrieved only from the mostabundant spots. For instance, the group of seven spots of23 kDa, more abundant in adults (group D, Fig. 1), pre-sented the same N-terminal sequence (TPEQQRYVEL-LXVVD), where X stands for an undetermined amino acid.This sequence was 73 % identical to a fragment of the 23kDa metalloproteinase bothrolysin [sptrembl: P20416] (1TPEHQRYIELFLVVD 15) and to an internal sequence ofthe 50 kDa metalloproteinase bothrostatin [sptrembl:Q98SP2] (188 TPEHQRYIELFLVVD 202), both from B.jararaca. However, Edman sequencing failed to identify a52 kDa polypeptide that was detected as the most abun-dant spot in the 2-DE map of adult venom (group A, Fig.1).

In-gel digests of non-identified spots were further ana-lyzed by peptide mass fingerprinting (PMF). Only a fewfull length sequences of B. atrox proteins are currentlyavailable in a database, and therefore it is not surprisingthat PMF searches did not provide significant scores forany of the 2-DE spots present in the gels. However, thisapproach allowed us to group spots whose peptide massfingerprints were similar. Based on the PMF data, all spotswere arranged in 27 groups. The 2-DE maps of juveniles,sub-adults and adults showing the groups containingspots with similar PMF spectra are shown in Fig. 1. Allgroups were found in juvenile venom gels while adult gelsdisplayed only 13 groups, revealing significant changes ofB. atrox venom proteome during ontogenetic develop-ment.

One spot of each group was submitted to protein sequenc-ing by tandem mass spectrometry followed by Mascot andMS BLAST database searches (Fig. 2), which enabled




Proteome maps of B. atrox venom from juveniles, sub-adults and adults. Spots displaying similar peptide mass fingerprints were grouped as explained in Results sectionFigure 1Proteome maps of B. atrox venom from juveniles, sub-adults and adults. Spots displaying similar peptide mass fingerprints were grouped as explained in Results section. A summary of the protein classes identified in each group is also shown.


Table 1: Protein groups identified in B. atrox venom proteome. Groups from A to ∆ comprise spots that displayed similar peptide mass fingerprints shown in Fig 3. pI range, molecular mass (MM) and relative expression were determined by computational analyses of the 2-DE gels. X: unknown amino acid; B: cleavage site; J: juveniles; S: sub-adults, A: adults.

Group Protein Class Best Match sptrembl # Organism

Peptide sequences pI range MM (kDa) Relative expression

A Metalloproteinase BothropasinO93523

Bothrops jararaca

BITVKPDVDYTLNSFAEWRBASMSECDPAEHCTGQSSECPADVFHKBMYELANIVNEIFRBKIPCAPEDVKBKTDLLTRBGMVLPGTKBXXFQDVYEAEDSCFKQYNPFRYLEFLLVVDQLLNKBYNPFR

5.0 52 J = S = A

B L-aminooxidase

L-amino oxidase IQ6TGQ9

Bothrops jararacussu

BXXGQLYEESLQKBLFLTMNKBXXTVTYQAVMKBIKFEPPLPPKKBRFDEIVGGMDKBHDDIFAYEKBXXWYANLGPMRLPEKBFWEDDGIHGGKBETDYEEFLEIAK

5.9 –6.2 55 – 61 J = S = A

C Metalloproteinase BothropasinO93523

Bothrops jararaca

BXXVEVGEECDCGSPRBXXGTECQAADMADLCTGRBLYCVDSAVNGRBLYCVDSSPANKBGMVLPGTKBKIPCAPEDVKBYNPFRBXXFQDVYEAEDSCFK

7.1–7.5 40 – 51 J>S>A

D Metalloproteinase MetalloproteinaseQ8QG89

Bothrops insularis

BTLDSFGEWR-BTLDSFEGWRBTLDSFWWRBTLDSFWEGRBTLDSFWGERBYVDLFLVVDHGVLDNKBYVDLFLVVDHGMFMK-BYVDLFLVVDHGXXXKBDLINVQQDSRBENPQCILNKR

5.3 – 6.4 23–24 J<S<A

E Serine proteinase BilineobinQ9PSN3

Agkistrodon bilineatus

BVVGGDECNINEHRBSLPSSPPSVWSASKBSLPSSPPSVXXSASKBDIMLIRBVMGWGSLSSPKBFLCNPRBFLCGGPRBIFLTCTK

6.3 –6.4 28 – 29 J = S = A

F CRISP AblominQ8JI40

Agkistrodon halys blomhoffi

BSVNPTASNMLKBMEWYPEAAANAERBKPEIQNEIVDLHNSLRBSGPPCGDCPSACDDGLCTNPCTKAGCAAAYCPSSSYKBXXDFDSESPR

7.1 –8.1 28 – 29 J>S = A

G Phospholipase A2 Phospholipase A2 Q7ZTA7

Crotalus viridis

BVAVLCFRFVHDCCYGKBMDLYTLHR-BMDLYTYDK

8.1 16 J = S = A



H C-type lectin Botrocetin β chainP22030

Bothrops jararaca

Platelet glycoproteinIB-binding protein

Q9PSM5Bothrops jararaca

Phospholipase A2 Q8QG87

Bothrops insularis

BDCPSGWSSYEGSCYKFVVTEYECVASKBXXSFEWSDGTDLSYKBXXTQLAYVVCK-BLAYVVCEAQR-BLAYVVCKBTCLGLEEDSGFRBSLTSPMLKDNQWLSFPCTRBXXNAWLGLWEGKBXXSFEWSDGTDLTYKBXXWADAESLCALQRBTCLGLEEDSGFRKBFCTLQQRBLLQLLQRBEEADFVRBNFVCEFQABWWIIPCTRBVAAICFRBVAATCFRBYWLYGAK

4.8 17 J = S<A

I C-type lectin Botrocetin α chainP22029

Bothrops jararaca

BDCPSGWSSYEGNCYKBLYSGEADFVGDLVTKBWSDGSSVSYENVVERBMNWADAERBKCFALEKBGGHLVSIKBCFALEKBNPFVCKBFFQQK

5.3 15 J = S<A

J Phospholipase A2 Myotoxin IP20474

Bothrops asper

BAAAVCFRBSLIEFAKBMILEETKBKSGVIICGEGTPCEKBXXAYPDLFCKBLYSGEADFVVKBVAVLCFRBAAGLCGFRBVTGVPTYKBSGVIICGE

7.8 17 J = S<A

K Phospholipase A2 Myotoxin IIIQ9PVE3

Bothrops asper

BXXVCDENNPCLKBXXVCDENNPPGR-VCDENNPCLKBYFAYPDLFCKVTSYQYBMILQETGKNPVTSYGAYGCNCGVLGRBELCECDKAVAICLRBAVAICLRBYSYSWK

7.8 17 J = S<A

L Metalloproteinase BerythractivaseQ8UVG0Bothrops

erythromelas

BLTPGSQCADGLCCDQCRBKYVEFVVVLDHGMYKBVPLTGLELWSDRBLYCFLYSSKVVFEPFK-VVQHQVRBVPLTXVLDHRFKKIPCAPEDVK

5.0 –5.9 61 – 66 J>S>A

M Metalloproteinase BOJUMET IIQ7T1T5

Bothrops jararacusu

BETVLLNRBYLIDNRPPCILNIPLRBFALVGLEMWSNRBFALVGLDMGWSNRBSSDLGMVDLASKVQGPLGNTLTCMPTDTDFDGTLLGLAWRTDTDFDGTLLGLAWRGQSADCPTDDLQR

5.4 –5.7 51–53 J>S>A

Table 1: Protein groups identified in B. atrox venom proteome. Groups from A to ∆ comprise spots that displayed similar peptide mass fingerprints shown in Fig 3. pI range, molecular mass (MM) and relative expression were determined by computational analyses of the 2-DE gels. X: unknown amino acid; B: cleavage site; J: juveniles; S: sub-adults, A: adults. (Continued)



N Serine proteinase Serine proteinaseQ7T229

Bothrops jararacusu

BLVGGDECNINEHRBIMGWGTLSPTKBFLCNPRBVSDYTEWLK

5.0 –5.3 29 – 30 J>S = A

O Metalloproteinase MetalloproteinaseQ8AWX7

Agkistrodon halys palas

BYIELVIVADHRBSVGIVRDYRBSVANDDEVIRYPK

5.2 –5.4 36 – 37 J>S>A

P Metalloproteinase Factor X activatorheavy chain

Q7T046Vipera lebetina

BLYETVNALNVLCRBYSVGLVQDYRBYIELVIVADHRBLNLNPDEQR

5.8 –6.2 38 – 40 J>S>A

Q Serine proteinase Serine proteinaseQ8QG86

Bothrops insularis

BFLAFLYPGRBIYLGIHARBDIMLIRBLHEPALYTKBLQGLVSDHRBSVANDDP-BSVANDDEE

6.3 –7.4 32 – 34 J = S>A

R Serine proteinase Catroxase IIQ8QHK2

Crotalus atrox

BLDDVLDQDLGBTLCAGILEGGKBAAYPELPATSRBAAYPERFTSRBLPSNPPW-BLPSNPPWHRBLPSNPPXXXR

6.5 29 J = S>A

S C-type lectin Alboaggregin Asubunit 2P81112

Trimeresurus albolabris

WADAERSYENWTEAELKBTCLGLEEDSGFRBTCLALEEDSGFRFCAGYLENKBTPLNLNCRBXXLNLNCRBXXTADAER

4.8 17 J>S = A

T C-type lectin MucrocetinQ6TPG9

Trimeresurus mucrosquamatus

BDCPSGWSSYEGSCYKBFCTQQQTNHLVSFQSRBXXCQFVVTEYPSFQSKBXXSFEWSDGTDLSYKBXXTQLAYVVCKBTTENQWWSRBXXLNLNCRBLFLQQNKBLTSPLLRBMNWEDAEK

4.8 15 J = S>A

U C-type lectin Alboagregrina AP81114

Trimeresurus albolabris

BDCPSDWSSYEGHCYRBVFNEPQNWADAEKBQSSEEADFVLKBSSEEADFVLKBXXLDQLLKBLFLQQNK

5.1–5.4 14 J = S>A

V C-type lectin Bothrocetin α chainP22029

Bothrops jararaca

BCFVLERBXXSDGSCVCYENLVERBEGFLTWRBSPMSPDTEEGKBXXCYENLVER

5.1–5.4 14 J = S>A

X VEGF Vascular Endothelialgrowth factor

Q90X24Bothrops insularis

CCTDESLECTATGKBLEVMKFTEHTNCECRBETLVSLLEEHPDEPSCVTALRBLFRAVLFNALR

5.6 –6.3 13 J>S>A

W C-type lectin Botrocetin α chainP22029

Bothrops jararaca

BDCPSGWSSYEGHCYRBXXWSDGSSVSYENLVERVSFQSDWTDFVVKBLVSFQSDWTDFVVKBLWADAERBNPFVCKBFCSEQAKBNIQSSDLYAWIGLR

5.9 –6.4 15 J = S>A




Y Phospholipase A2 Phospholipase A2 Q8QG87

Bothrops insularis

BYFSYGCYCGLGGLGQPR-GSYGCYCLGGLBXXFVHDCCYGKBXXXFVHDCCYGKBXXKDTYNLQYWLYQK-BXXKDTYNLKYWLYAGKBLTYNLQYWLYQK-BLTYNLKYWLYAAGKBYGEGLYQK-BYGEGLYAGKBVVTTCFRBVAVLCTRBQLCECDFVABXXCECDFVBXXLWQFGT

6.7 15 J = S>A

Z C-type lectine NGF

Platelet glycoproteinIB-binding protein

α chainQ9PSM6

Bothrops jararaca

Nerve growth factor

Q9DEZ9Crotalus durissus

terrificus

BQYFFETKBALTMEGNQASWRBIDTACVCVISRBXXALGQKBFIRPRBNPFVCKBFFQQKBDTPFECPSDWSTHRBXXSDGSCVCYENLVR

7.8 17 J = S>A

∆ Phospholipase A2 Myotoxin IIIQ9PVE3

Bothrops asper

BSYAAYGCNCGVLGRBMLLLETGKLPAKNLWQLGKVAVLCFRBAVAICLRBYSYSWKBYNYLKPFCKBTIVCGENNSCLK

6.4 –8.1 16 J = S>A





MS BLAST identification of a spot from group YFigure 2MS BLAST identification of a spot from group Y. Panel A: nanoES mass spectrum of the unseparated in-gel tryptic digest. Peaks of trypsin autolysis products are designated with "T". Peptide precursor ions, whose tandem mass spectra were acquired and interpreted are designated with corresponding m/z and charge (in parenthesis). Panel B: MS/MS spectrum of the precur-sor ion with m/z 744.77 (designated with asterisk in panel A). De novo sequencing was performed by considering mass differ-ences between the adjacent peaks in the series of fragments that belong to the ions containing C-terminal amino acid residue (y-ions), starting from high m/z region of the spectrum. The sequence shown in the panel was deduced by considering the most abundant fragment ions and was not necessarily correct; a few optional variants of interpretations based on the low abundant fragments and arriving to the typical tryptic C-terminus (K) were possible. All sequence candidates for used in a single MS BLAST search. Panel C: MS BLAST query that comprises all sequence proposals obtained by the interpretation of all tandem mass spectra were assembled in an arbitrary order and spaced by a minus (gap) symbol. B stands for a generic trypsin cleavage site (R or K) preceding peptide sequences and is introduced if it was possible to read out the sequence until the very N-termi-nus of the peptide. Panel D: The top confident hit of MS BLAST search. The search also reported a few homologous proteins from other species.

Relativeintensity,%

50

100

m/z750700650600550500450

*744.77( +2)

699.78( +2)

664.01(+3)

652.32(+3)

622.30(+3)

615.28( +3)

609.62

(+3)

594.30(+3)

559.29(+3)

545.62(+3) T T

498.00(+4)

479.26(+2)

450.71(+2)

441.72(+2)

432.73

(+2)

200 300 400 500 600 700 800 900 1000 1100 1200

Relativeintensity,%

100

50

744.77 (+2)

FVHDC CYG[K]

m/z

m/z 652(+2): -BYFSYGCYCGLGGLGQPR-GSYGCYCLGGL m/z 699(+2): -BXXFVHDCCYGK m/z 744(+2): -BXXXFVHDCCYGK m/z 545(+3): -BXXKDTYNLQYWLYQK-BXXKDTYNLKYWLYAGK m/z 664(+2): -BLTYNLQYWLYQK-BLTYNLKYWLYAGK m/z 479(+2): -BYGEGLYQK-BYGEGLYAGK m/z 441(+2): -BVVTTCFR m/z 432(+2): -BVAVLCFR m/z 615(+3): -BQLCECDFVA m/z 609(+3): -BXXCECDFV m/z 594(+3): -BXXLWQFGT

Q8QG87 Phospholipase A2 BITP01A(EC 3.1.1.4), Length = 138

Total Score: 391

Score = 106

Query: 2 BYFSYGCYCGLGGLGQPR 19

+YF YGCYCG GG+GQPR

Sbjct: 36 KYFYYGCYCGWGGIGQPR 53

Score = 103

Query: 87 BXXKDTYNLKYWLYGAK 103

+ KDTY+ KYWLYGAK

Sbjct: 113 RDNKDTYDMKYWLYGAK 129

Score = 81

Query: 43 BXXFVHDCCYGK 54

+ FVHDCCYGK

Sbjct: 58 RCCFVHDCCYGK 69

Score = 58

Query: 169 BQLCECDFVA 178

+Q+CECD VA

Sbjct: 99 KQICECDRVA 108

Score = 43

Query: 151 BVVTTCFR 158

+V TCFR

Sbjct: 106 RVAATCFR 113

A B

C D


either to identify the protein, or to assign it to a class ofhighly homologous proteins. In this way, metalloprotein-ases, L-amino oxidases, serine proteinases, cysteine-richsecretory proteins (CRISPs), phospholipases A2, lectinsand growth factors were identified (Table 1).

Given that very few sequences of B. atrox proteins areavailable in a sequence database, the best matches of MSBLAST database searches corresponded to proteins foundin venoms of other snakes, mostly of the Bothrops genus(Table 1). Since several snake venom proteins share highsequence similarity and peptides analyzed by MS/MScover only a small fraction of their sequences, it was notpossible to unequivocally determine the protein homo-logues from the sequenced species. MS/MS analysis of thespot with apparent MW of 52 kDa (group A, Fig. 1 andTable 1) that was present in similar quantities in adult,sub-adult and juvenile gels is presented here as an exam-ple. MS/MS sequencing identified it as a member of the P-III class Zn-metalloproteinase. The best matches corre-sponded to the jararhagin and bothropasin, high molecu-lar mass metalloproteinases from B. jararaca, which shareapproximately 95% of sequence identity. One of thedetermined sequences (KINPFR) is present in bothropa-sin, but not in jararhagin and another (BMYELANIV-NEIFR) shares 100% identity with jararhagin only,because there is a substitution (F→ L) in bothropasin. Theremaining peptide sequences (Table 1) are present in bothbothropasin and jararhagin. Therefore, the 52 kDa spot isunequivocally related to a metalloproteinase from the P-III class that is homologous, albeit is different from bothbothropasin and jararhagin.

The proteins from group C, whose molecular masses aresimilar to those of group A, were identified as metallopro-teinases of the P-III class, albeit having higher pI values(7.1–7.5 comparing to pI 5 of group A). They share twopeptide sequences (BKIPCAPEDVK and BGMVLPGTK)with the polypeptides from group A. Two other sequences

(BXXVEVGEECDCGSPR and BLYCCVDSSPANK)matched bothropasin only partially and another sequence(BXXGTECQAA) occurs in metalloproteinases from otherspecies of vipers. Differently from group A, the group Cproteins are much more abundant in juveniles than adultsas shown in Fig. 3.

Group D, one of the most prominent groups of spots inadult gels, comprised several isoforms of approximately23 kDa and pI range between 5.3 and 6.4 (Fig 3). On theother hand, only two of these spots (in very low amounts)were detectable on silver stained gels of juvenile venom.Comparison of the three gels suggested that the concen-tration of 23 kDa isoforms increased during ontogeneticdevelopment (Fig. 3). N-terminal sequences of these pro-teins were determined by Edman degradation and theproteins were found to be homologous to bothrolysinand bothrostatin metalloproteinases (see above). MSBLAST searches with a query composed of peptidesequence proposals obtained by the de novo interpretationof MS/MS spectra produced as best hit a P-II class metal-loproteinase from B. insularis. Snake venom metallopro-teinases with molecular masses 23–25 kDa, which arecomposed by a sole metalloproteinase domain, are usu-ally assigned to the P-I class [4,6]. Some P-I class proteinsare produced by proteolytic processing of P-II metallopro-teinases, which are larger and contain a desintegrin-likedomain besides the metalloproteinase domain [7]. There-fore, proteolytic processing could explain the high simi-larity of proteins from group D with P-II classmetalloproteinases. The spots from group D probably cor-respond to the 23 kDa polypeptide previously purifiedfrom B. atrox venom that constituted a single band in SDS-PAGE [2]. Further ESI-MS experiments suggested thatthere were at least three isoforms of this protein in the B.atrox venom [2]. Here, 2-DE gels were able to resolve atleast seven isoforms in the venom of adults. Image analy-ses of the gels showed a remarkable increase in the vol-ume of group D spots from juveniles to adults.

The group L comprised at least seven isoforms that wereabundant in juvenile, less abundant in sub-adult andundetectable in adult venoms. They were homologous toberythractivase, a 78 kDa P-III class metalloproteinasefound in B. erythromelas that is a protrombine activatorthat possess a high pro-coagulant activity [8].

The proteins from group M were also identified as metal-loproteinases and were not detected in adults. These pro-teins were homologous to BOJUMET II from B. jararacusuas well as to berythractivase. Although the proteins fromgroups L and M were both homologous to berythracti-vase, PMF and MS/MS data indicated that they were, infact, different proteins rather than post-translationallymodified forms of the same gene.

Zoomed gels showing examples of differential expression of proteins of groups D and C upon ontogenetic developmentFigure 3Zoomed gels showing examples of differential expression of proteins of groups D and C upon ontogenetic development.



Another group of spots (B) with molecular mass range of55–61 kDa contained several isoforms present in similaramounts in the three different venom samples. Theirsequences were homologous to apoxin I, a L-amino oxi-dase (LAO) from Crotalus adamanteus. LAO are majorcomponents of snake venoms that cause cell death byapoptosis [9].

The proteins from the groups E, N, Q and R were identi-fied as serine proteinases. The snake venom serine protei-nases possess thrombin-like activity and several of themhave been isolated from bothropic venoms [4].

Proteins belonging to the class of Cysteine Rich SecretoryProteins (CRISP) were identified in group F and displayedsimilar expression in the three developmental stages ana-lyzed. The CRISPs are found in epididimus and granularcells of mammals and seem to act in cell maturation ofspermatozoa and cells from immune system, though theexact function of these proteins is unknown [10]. Mem-bers of CRISP family isolated from snake venoms, such asablomin and trifilin, are responsible for blocking ofsmooth muscles contractions induced by depolarization.Immunological screening using anti-triflin antiserumidentified CRISPs in different snake venoms, although thecross-reactivity was relatively low for the only member ofthe Bothrops (B. jararaca) genus tested [10].

In the low molecular mass range (12–15 kDa), severalphospholipases A2 (PLA2) and C-type lectins were found.Within these groups, we identified at least four variantsequences of the same peptide stretch present in PLA2(VAVLCFR, AAAVCFR, VAATCFR and VAVLYSR) indicat-ing a high degree of polymorphism for these enzymes.Other examples of polymorphic peptide sequences wereobserved in groups W (more expressed in juveniles andsub-adults) and I (adult specific). MS BLAST searches pro-duced as best hits the alpha chain of the C-type lectinbothrocetin, although the sequence of their peptides andthe PMF analysis showed that they contained differentproteins. C-type lectins were also identified as the majorcomponent in groups U and V. Interestingly, groups H(adults) and S (juveniles), despite having similar pI,molecular weight and some peptide sequences, actuallycontained different C-type lectins.

Proteins homologous to vascular endothelial growth fac-tor (VEGF) from Bothrops insularis were detected in groupX and their abundance decreased during ontogeneticdevelopment (Fig. 1). VEGFs from snake venoms areknown to enhance the vascular permeability and play animportant role in the initial stages of envenoming by Both-rops. It is assumed that snake venom VEGFs, dstimulatethe distribution of the venom favouring both local andsystemic actions [11].

Group Z displayed only one detectable spot that waspresent in juveniles and absent from adults. It was identi-fied as a mixture of a nerve growth factor (NGF) and a C-type lectin. NGF belongs to a family of neurotrophic fac-tors, which are endogenous soluble proteins regulatingsurvival, growth morphological plasticity or synthesis ofproteins for differentiated functions of neurons. In addi-tion, there is increasing evidence that NGF activities arenot restricted to the nervous system, but also affect non-neuronal cells, especially those of haematopoietic stemcell origin. NGF was previously described in the threemain families of venomous snakes (Viperidae, Crotalidaeand Elapidae). The ubiquitous presence of NGF in snakevenoms suggests a toxinological importance of that pro-tein in a sense of direct toxic action, indirect toxic actionor an activity in the context of prey digestion [12].

DiscussionThe proteome composition of snake venom alters withage and therefore the developmental stage of the organ-ism, which donated the specimen, should be taken intoaccount. To this end, we performed a comparative analy-sis of B. atrox venom proteome in three stages of maturity.To our knowledge, this is the first proteome analysis ofsnake venoms associated with the ontogenetic develop-ment, although other proteomic studies on snake venomhave been published previously [13,14].

In a typical proteomic routine, proteins are separated by2-DE, visualized by silver or Coomassie blue staining [15]and protein spots in-gel digested with trypsin [16]. Analiquot of the digest is subjected to PMF by MALDI-TOFmass spectrometry. If no conclusive identification isachieved, tryptic peptides are then extracted from the gelmatrix and sequenced by tandem mass spectrometry [17].However, in both protein identification approaches it isrequired that the exact sequence of the analyzed protein isavailable in a database. Nevertheless, only a small numberof B. atrox proteins are currently available in a sequencedatabase and therefore the scope of protein identificationwas very limited. To address this issue, we complementedconventional protein identification methods, which arebased on the exact matching of tandem mass spectra todatabase sequences, with sequence-similarity searches.The latter approach enabled confident identification ofunknown proteins that only distantly related to knownproteins from other species [18-20]. Using a combinationof stringent and sequence-similarity database searches, weidentified all major components of venoms and mappedout changes in the abundance of individual protein com-ponents in ontogenetically altered proteomes.

The natural polymorphism of the protein sequences,together with the absence of a complete and annotatedsnake genome, which could be used as a referenceid not



allow the unambiguous assignment of the most relatedprotein homologues due to the low sequence coverage.However, the group-specific assignment, based on thepeptide sequences, was always reliable.

Based on peptide mass fingerprinting analysis we classi-fied the spots of the 2-DE maps in 27 groups. Most groupsand those with larger number of spots corresponded toproteinases (metallo and serine proteinases). This resultagrees with the fact that venoms from Bothrops species arehaemotoxic and promote haemorrhaging primary toextensive local swelling and necrosis [14].

The proteome maps also enabled the identification ofnew groups of potential ontogenetic molecular markers.For instance, we found that some groups of proteinsincluding P-III class metalloproteinases (L, M, O and P),serine proteinases (N, Q and R) are more abundant injuvenile specimens, while metalloproteinases from classP-I (group D) are more expressed in adults.

Overall, more groups of proteins were identified in theproteome maps from juveniles, than in adults. Proteomemaps from sub-adults contained spots from both juve-niles and adults, along with a few stage-specific spots. Pre-vious work suggested that the B. atrox venoms from youngindividuals trigger more potent biological effects thanadult venoms [2,5]. The decrease in hemorrhagic activityin B. atrox venoms during ontogenetic development maybe explained by the lower levels of Zn-metalloproteinasesof P-III class in adults comparing to juveniles. On theother hand, the higher concentration of berythractivase, apro-thrombin activator, may reflect the higher coagulantactivity of juvenile venoms. The effect of VEGFs, NGFs andCRISPs, all more expressed in young specimens, certainlyalso contribute to the higher pharmacological activities ofthe juvenile venom.

The diversity in the protein composition and biologicalactivity of snake venom during growth may be related toadaptation by evolutionary processes to the type and sizeof the prey [21-24]. Young Bothrops snakes preferentiallyeat amphibians, lizards, birds, and shift to mammalswhen they become adults [25]. Therefore, the qualitativeand quantitative changes in the B. atrox venom proteomeare most likely related to the survival of the snake by preyadaptation.

ConclusionWe have established proteome maps for the venom of B.atrox in three different developmental stages, i.e. juvenile,sub-adult and adult. Analysis of the proteome maps con-firmed that B. atrox venom proteome alters significantlywith aging of the animal. Moreover, we have verified theexistence of stage specific and differentially expressed

polypeptides that may be responsible for the diverse activ-ities of the venom in each developmental stage analyzed.

The proteome of any biological sample is expected todynamically change in response to external stimuli. Thechanges in the B. atrox venom proteome reported herereinforce the need to intensify the studies on organisms indifferent stages of maturation. This procedure may lead tothe discovery of a wider group of molecules of biotechno-logical and medical interest, as well as to better under-standing of important biological traits of the organismduring its ontogenetic development.

Finally, intra-species variations in the composition ofsnake venom during ontogenetic development promptsfurther studies of the relationship between symptomatol-ogy of snake bite accidents in humans with the venomcomposition as well as the use of venoms from individualspecimen of various ages for the production of antiven-oms.

MethodsSnake venomsVenoms were obtained from wild Bothrops atrox speci-mens with no known litter relationships, captured inManaus region (Amazonas State, Brazil), and maintainedin the Herpetarium of the Gerência de Animais Peçonhen-tos-IMT-AM, Manaus. Classification of B. atrox snake spec-imens was done based on the total lenght of wildspecimens according to Martins and Oliveira (1998) [26].Therefore, three size classes were defined: juveniles (≤ 40cm), sub-adults (> 40 – 70 cm), and adults (> 70 cm).Twenty days after the snakes were captured and beforetheir first feeding in captivity, the venoms were manuallymilked by massaging the venom glands of specimenslonger that 50 cm, and with the aid of a Pasteur pipette onsnakes smaller than 50 cm. A single extraction was per-formed for each specimen. Three types of pooled venomsamples were prepared, juvenile, comprising venoms ofeight specimens, sub-adult, corresponding to twenty fivespecimens and adult, a mixture of venoms of twelve spec-imens. The pooled venom samples were centrifuged, fil-tered, lyophilized, weighted and stored at -20°C.

Two-dimensional gel electrophoresisFreeze dried pooled venom samples (100 µg for silverstained gels and 300 µg for Coomassie blue stained gels)were solubilised in 370 µL of 2-DE sample buffer (7Murea, 2M thiourea, 1% DTT, 2% Triton X-100, 0.5% Phar-malyte 3–10) containing proteinase inhibitors (100 µMPMSF, 1 µM pepstatin A and 5 mM EDTA) and applied to18 cm IPG gel strips (GE Healthcare, Upsala, Sweden)containing linear or non-linear 3–10 pH gradient by in-gel sample rehydration [27]. After 12 h of rehydration inthe sample solution, isoelectric focusing (IEF) was carried



out at 20°C using an IPGphor unit (GE Healthcare) inthree successive steps: 500 Vh, 1000 Vh and 32000 Vh. Forreduction and alkylation the IPG gel strips were soaked for30 min in a solution containing 50 mM Tris pH 8.8, 6 Murea, 30% glycerol, 2% SDS and 125 mM DTT and for anadditional 30 min in the same buffer containing 125 mMiodoacetamide and bromophenol blue instead of DTT.SDS-PAGE was performed on 10–20% T polyacrylamidegradient gels on a Protean II system (Bio Rad, Hercules,CA, USA) connected to a Multitemp II cooling bath (GEHealthcare). Electrophoresis was carried out at constantcurrent (25 mAmp per gel) at 20°C until the dye frontreached the lower end of the gel.

2-DE gels containing 100 µg of venom were silver stained[28] and submitted to image analysis. Alternatively, 2-DEgels containing 300 µg of venom were stained with 0.1%(w/v) Coomassie Blue in 40% (v/v) methanol, 10% (v/v)acetic acid and distained with the same solution withoutCoomassie Blue.

Image analysisSilver stained gels were scanned with a SHARP JX-330scanner (Tokyo, Japan) at 300 dpi resolution and the tiffimages generated were analyzed with Image Master 2DElite software (GE Healthcare). Spot detection and spotmatching were performed in automated mode. Spot vol-umes were acquired without background subtraction andnormalized using the total intensity of the detected spots.

Protein digestionCoomassie stained spots were excised from the prepara-tive gel and in-gel digested with trypsin as described in[15].

Peptide mass fingerprintingProtein digests were first subjected to peptide mass finger-printing by matrix-assisted laser desorption/ionization ona Bruker Reflex IV time-of-flight (MALDI-TOF) mass spec-trometer equipped with Scout 384 ion source. Probeswere prepared by dried-droplet method as described pre-viously [29]. Briefly, 1 µL aliquot of the digest was mixedon the surface of AnchorChip™ 384/600 targets (BrukerDaltonics, Germany) with a saturated solution of matrix(α-cyano-4-hydroxycinnamic acid) in 1: 2 (v/v) solutionof 2.5% aqueous TFA: acetonitrile. The mixture wasallowed to dry at room temperature and the entire targetwas washed with 5 % formic acid.

Spots whose peptide mass fingerprints were similar, i.e.shared more than 75% of peaks with the relative intensityof more than 20 % within the mass accuracy of +/- 100ppm, after removal of peaks of trypsin autolysis productsand known keratin contaminants, were grouped as shownin Fig 1.

Sequencing by nanoelectrospray tandem mass spectrometryIf peptide mass fingerprinting did not identify the protein,peptides were extracted from the gel pieces with 5 % for-mic acid and acetonitrile and the extracts were pooledtogether and dried down in a vacuum centrifuge. Thedigests were taken up in 5 % formic acid and analyzed bynanoelectrospray tandem mass spectrometry on a hybridquadrupole time-of-flight instrument QSTAR Pulsar i(MDS Sciex, Canada) as previously described [30,31].

Database searchingPeptide mass fingerprints were used for database search-ing using Mascot software (Matrix Science Ltd, UK)against the MSDB database (updated May 15, 2005; con-taining 2011425 protein sequence entries) downloadedfrom NCBI. Mass tolerance was set to 100 ppm, spectrawere calibrated externally and no restrictions wereimposed on protein molecular mass or phylogenetic line-age. Uninterpreted tandem mass spectra were firstsearched by Mascot against the above database to identifyproteins with tryptic peptides identical to databaseentries. Precursor mass tolerance was set at 0.1 Da andfragment ion mass tolerance at 0.05 Da. Hits were consid-ered significant if their protein score exceeded the thresh-old score calculated by Mascot software assuming p <0.05. Matched MS/MS spectra were further manuallyinspected considering the correlation of y-, b- and a- frag-ment ions [32] to corresponding m/z calculated from thepeptide sequences. If still no identification was achieved,tandem mass spectra were interpreted de novo using Bio-Analyst QS software as previously described [31]. Multiplesequence candidates were allowed per each interpretedtandem mass spectrum and partial peptide sequenceswere included into the search string. All candidatesequences were merged into a single search string and MSBLAST searches were performed against a non-redundantprotein database (nrdb) at http://genetics.bwh.harvard.edu/msblast/ or http://dove.embl-heidelberg.de/Blast2/msblast.html under default settings. Parsing scriptoperating at the MS BLAST web site was applied to identifyand colour code statistically confident hits according toMS BLAST scoring scheme [20]. According to the selectedMS BLAST threshold scores of statistical confidence, theexpected rate of false positive identification was lowerthan 1 %.

Abbreviations2-DE: two-dimensional gel electrophoresis, IPG: immobi-lized pH gradient, CRISP: Cysteine Rich Secretory Pro-teins, PLA2: phospholipase A2,VEGF: vascular endothelialgrowth factor, NGF: nerve growth factor, IEF: isoelectricfocusing, DTT: dithiotreitol, PMSF: phenylmethyl sulfo-nyl fluoride, EDTA: ethylenediaminetetraacetic acid.


http://genetics.bwh.harvard.edu/msblast/

http://genetics.bwh.harvard.edu/msblast/

http://dove.embl-heidelberg.de/Blast2/msblast.html

http://dove.embl-heidelberg.de/Blast2/msblast.html


Competing interestsThe author(s) declare that they have no competing inter-ests.

Authors' contributionsRAPG- Participated in 2D-PAGE, protein blotting, dataanalysis and writing of the manuscript.

Anna S – Peptide mass fingerprinting, MS/MS analysisand database searching.

Andrej S- Database searching and data analysis. Partici-pated in the writing of the manuscript

JLLP- Animal collection, venom sample preparation andgeneral discussion of results

J. P- Optimization of 2D-PAGE conditions and computa-tional image analysis

MVS- Participated in the design of the study, coordinationand writing.

CAOR- Experimental design, data analysis and coordina-tion. Preparation of the final version of the manuscript.

All authors read and approved the final manuscript.

AcknowledgementsThis work was supported by PADCT /MCT and FINEP (CT-INFRA). A stu-dentship from CAPES was awarded to Rafael A. Pontes Guércio.

References1. Hoge AR, Romao Hoge SAWL: Poisonous snakes of the world.

Part I. Check list of the pit vipers viperoidea, crotalinae.Memorias do Instituto Butantã 1978, 42:79-310.

2. López-Lozano JL, Sousa MV, Ricart CAO, Cháves-Olortegui C,Sanchez EF, Muniz EG, Bührnheim PF, Morhy L: Ontogenetic vari-ation of metalloproteinases and plasma coagulant activity invenoms of wild Bothrops atrox specimens from Amazonianrain forest. Toxicon 2002, 40:997-1006.

3. Kamiguti AS, Hay CRM, Zuzel M: Inhibition of collagen-inducedplatelet aggregation as the result of cleavage of α2β1-integrin by the snake venom metalloproteinase Jararhagin.Biochem J 1996, 320:635-641.

4. Matsui T, Fujimura Y, Titani K: Snake venom proteases affectinghemostasis and thrombosis. Biochim et Biophys Acta 2000,1477:146-156.

5. Saldarriaga MM, Otero R, Nuñez V, Toro MF, Díaz A, Gutierrez JM:Ontogenetic variability of Bothrops atrox and Bothrops aspersnake venoms from Colombia. Toxicon 2003, 42:405-411.

6. Hati R, Mitra P, Sarker S, Bhattacharyya KK: Snake venom hemor-rhagins. Critical Reviews in Toxicology 1999, 29:1-19.

7. Takeya H, Nishida S, Miyata T, Kawada SI, Saisaka Y, Morita T, Iwan-aga S: Coagulation factor X activating enzyme from Russell'sviper venom (RVV-X). J Biol Chem 1992, 267:14109-14417.

8. Silva MB, Schattner M, Ramos CRR, Junqueira-De-Azevedo ILM,Guarnieri MC, Lazzari MA, Sampaio CAM, Pozner RG, Ventura JS, HoPL, Chudzinski-Tavassi AM: A prothrombin activator from Both-rops erythromelas (jararaca-da-seca) snake venom: charac-terization and molecular cloning. Biochem J 2003, 369:129-139.

9. Stábeli RG, Oliveira EB: Isolation and partial characterization ofthe L-amino acid oxidase from Bothrops alternatus snake

venom. Journal of Venomous Animal Toxins Including Tropical Diseases2003, 9:420.

10. Yamazaki Y, Hyodo F, Morita T: Wide distribution of cysteine-rich secretory proteins in snake venoms: Isolation and clon-ing of novel snake venom cysteine-rich secretory proteins.Arch Biochem Biophys 2003, 412:133-141.

11. Junqueira De Azevedo ILM, Farsky SHP, Oliveira ML, Ho PL: Molec-ular cloning and expression of a functional snake venom vas-cular endothelium growth factor (VEGF) from the Bothropsinsularis p it viper. J Biol Chem 2001, 276:39836-39842.

12. Kostiza T, Meier J: Nerve growth factors from snake venoms:chemical properties, mode of action and biological signifi-cance. Toxicon 1996, 34:787-806.

13. Serrano SMT, Shannon JD, Wang D, Camargo ACM, Fox JWA: mul-tifaceted analysis of viperid snake venoms by two-dimen-sional gel electrophoresis: An approach to understandingvenom proteomics. Proteomics 2005, 5:501-510.

14. Li ST, Wang JQ, Zhang XM, Ren Y, Wang N, Zhao K, Chen XS, ZhaoCF, Li XL, Shao JM, Yin JN, West MB, Xu NZ, Liu SQ: Proteomiccharacterization of two snake venoms: Naja naja atra andAgkistrodon halys. Biochem J 2004, 384:119-127.

15. Shevchenko A, Wilm M, Vorm O, Mann M: Mass spectrometricsequencing of proteins from silver-stained polyacrylamidegels. Anal Chem 1996, 68:850-858.

16. Havlis J, Thomas H, Sebela M, Shevchenko A: Fast-response pro-teomics by accelerated in-gel digestion of proteins. Anal Chem2003, 75:1300-1306.

17. Shevchenko A, Jensen ON, Podtelejnikov AV, Sagliocco F, Wilm M,Vorm O, Mortensen P, Shevchenko A, Boucherie H, Mann M: Link-ing genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensionalgels. Proc Natl Acad Sci USA 1996, 93:14440-14445.

18. Liska AJ, Shevchenko A: Expanding organismal scope of pro-teomics: cross-species protein identification by mass spec-trometry and its implications. Proteomics 2003, 3:19-28.

19. Shevchenko A, Sunyaev S, Loboda A, Bork P, Ens W, Standing KG:Charting the proteomes of organisms with unsequencedgenomes by MALDI-quadrupole time-of-flight mass spec-trometry and BLAST homology searching. Anal Chem 2001,73:1917-1926.

20. Habermann B, Oegema J, Sunyaev S, Shevchenko A: The power andthe limitations of cross-species protein identification bymass spectrometry-driven sequence similarity searches. MolCell Proteomics 2004, 3:238-249.

21. Theakston RDG, Reid HA: Development of simple standardassays procedures for the characterization of snake venoms.Bull World Health Organization 1983, 61:949-956.

22. Martins M, Gordo M: Bothrops atrox (common lancehead) diet.Herpetology Reviews 1993, 24:151-152.

23. Mackessy SP: Venom ontogeny in the pacific rattlesnakes Cro-talus viridis viridis and C. v. oreganus. Copeia 1988, 1:92-101.

24. Andrade DV, Abe AS: Relationship of venom ontogeny and dietin Bothrops Herpetologica. 1999, 55:200-204.

25. Daltry JC, Wuster W, Thorpe RS: Diet and snake evolution.Nature 1996, 379:537-540.

26. Martins M, Oliveira ME: Natural history of snakes in forest ofthe Manaus region, Central Amazonia, Brazil. Herpetol NatHistory 1998, 6:78-150.

27. Sanchez JC, Rouge V, Pisteur M, Ravier F, Tonella L, Moosmayer M,Wilkins MR, Hochstrasser DF: Improved and simplified in-gelsample application using reswelling of dry immobilized pHgradients. Electrophoresis 1997, 18:324-327.

28. Blum H, Bier H, Gross HJ: Improved silver staining of plant pro-teins, RNA and DNA in polyacrilamide gels. Electrophoresis1987, 8:93-95.

29. Thomas H, Havlis J, Peychl J, Shevchenko A: Dried-droplet probepreparation on AnchorChip trade mark targets for navigat-ing the acquisition of matrix-assisted laser desorption/ioniza-tion time-of-flight spectra by fluorescence of matrix/analytecrystals. Rapid Commun Mass Spectrom 2004, 18:923-930.

30. Shevchenko A, Chernushevich I, Wilm M, Mann M: "De novo "sequencing of peptides recovered from in-gel digested pro-teins by nanoelectrospray tandem mass spectrometry. MolBiotechnol 2002, 20:107-118.

31. Shevchenko A, Sunyaev S, Liska A, Bork P: Nanoelectrospray tan-dem mass spectrometry and sequence similarity searching






















































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for identification of proteins from organisms with unknowngenomes. Methods Mol Biol 2003, 211:221-234.

32. Biemann K: Contributions of mass spectrometry to peptideand protein structure. Biomed Environ Mass Spectrom 1988,16:99-111.







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